Lack of selectivity of chemotherapeutic agents is a major problem in cancer treatment. Because highly toxic compounds are used in cancer chemotherapy, it is typically associated with severe side effects. Drug concentrations that would completely eradicate the tumor cannot be reached because of dose-limiting side effects such as gastrointestinal tract and bone marrow toxicity. In addition, tumors can develop resistance against anticancer agents after prolonged treatment. In modern drug development, targeting of cytotoxic drugs to the tumor site can be considered one of the primary goals.
A promising approach to obtain selectivity for tumor cells or tumor tissue is to exploit the existence of tumor-associated enzymes. A relatively high level of tumor-specific enzyme can convert a pharmacologically inactive prodrug to the corresponding active parent drug in the vicinity of or inside the tumor. Via this concept a high concentration of toxic anticancer agent can be generated at the tumor site. All tumor cells may be killed if the dose is sufficiently high, which may decrease development of drug resistant tumor cells.
There are several enzymes that are present at elevated levels in certain tumor tissues. One example is the enzyme β-glucuronidase, which is liberated from certain necrotic tumor areas. Furthermore, several proteolytic enzymes have been shown to be associated with tumor invasion and metastasis. Several proteases, like for example the cathepsins and proteases from the urokinase-type plasminogen activator (u-PA) system are all involved in tumor metastasis. The serine protease plasmin plays a key role in tumor invasion and metastasis. The proteolytically active form of plasmin is formed from its inactive pro-enzyme form plasminogen by u-PA. The tumor-associated presence of plasmin can be exploited for targeting of plasmin-cleavable conjugates or prodrugs.
An enzyme can also be transported to the vicinity of or inside target cells or target tissue via antibody-directed enzyme prodrug therapy (ADEPT)1, polymer-directed enzyme prodrug therapy (PDEPT) or macromolecular-directed enzyme prodrug therapy (MDEPT)2, virus-directed enzyme prodrug therapy (VDEPT)3 or gene-directed enzyme prodrug therapy (GDEPT)4.
The technology of this invention relates to novel spacers (linkers) or spacer systems (linker systems) that can be inserted between a specifier (unit that can be cleaved or transformed) and leaving groups (for example parent drugs or detectable molecules). Furthermore, the invention relates to prodrugs and (bio)conjugates comprising a specifier, said novel spacers or spacer systems and multiple leaving groups, and to bifunctional linker systems comprising a (protected) specifier containing a reactive moiety that enables coupling to a targeting moiety on one side of the linker (system) and reactive moieties that enable coupling to multiple leaving groups (for example parent drugs or detectable molecules) on the other side of the linker (system). A great number of anticancer conjugates and prodiligs that have been developed in the past contain a self-eliminating connector or linker, also called self-elimination spacer. This spacer is incorporated between the specifier and the drug in order to facilitate enzymatic cleavage and so enhance the kinetics of drug release (as shown in FIG. 1). The specifier (which for example can be an oligopeptide substrate for a protease or for example a β-glucuronide substrate for β-glucuronidase) must be site-specifically removed or transformed, followed by a spontaneous spacer elimination to release the cytotoxic parent drug. Up to now self-elimination spacers have been implemented that release one drug molecule upon prodrug activation and subsequent spacer elimination. When the prodrugs and (bio-)conjugates containing multiple drug moieties are considered that have been reported thus far, an independent cleavage was necessary for each drug molecule to be released.
WO 98/13059 is a relevant disclosure describing a prodrug comprising an amino-terminal capped peptide covalently linked to a therapeutic drug through a self-eliminating spacer. In particular this document describes the use of p-aminobenzyl-oxycarbonyl (PABC) as self-elimninating spacer. The PABC electronic cascade spacer was already known from for instance Carl et al., J. Med. Chem., 1981, vol. 24, 479-480. Specifically the anticancer drugs doxorubicin, mitomycin C, paclitaxel and camptothecin coupled to PABC are described. A second self-eliminating spacer that is described is p-amino-bis(hydroxymethyl)styrene (BHMS), having the structure p-NH-Ph-CH═C(CH2O—)2, including the carbonyl groups the structure is p-NH-Ph-CH═C(CH2OCO—)2. It is noted that this spacer is described in this disclosure as a bis-carbamate, which teaches that two drug molecules are linked to this spacer via an amine functionality of the drug. The only drug moiety that is disclosed that is actually coupled to the BHMS spacer is doxorubicin. Doxorubicin is coupled via its sugar amino group resulting in a carbamate linkage between spacer and drug. Further it is stated that the spacer can bind two drug moieties. However, the document is silent on how many drug molecules are actually released.
Other systems that are loaded with multiple covalently bound bioactive molecules as end groups have been reported. Examples are systems that release doxorubicin after acidolysis of each hydrazone linker5, starlike HPMA copolymers containing doxorubicin6, or multi-loaded poly(ethylene glycol) prodrugs7. A doxorubicin-containing starlike HPMA copolymer with an antibody as the core has also been reported8. A number of recent publications have described the use of branched linkers in combination with antibody-containing prodrugs or bioconjugates with the aim of increasing the number of drugs bound per antibody9. However, to our knowledge, in each multi-loaded prodrug system reported so far, each single end group needs to be independently cleaved in order to release all end groups.
Thus there is a need for improved prodrugs or (bio-)conjugates in terms of (efficiency of) release of sufficient amounts of active drug in relation to the activation that is required at a desired site of action. In many cases, in prodrugs or (bio)conjugates, it is desirable to increase drug loading per targeting unit, in order to improve the efficacy of targeted compounds.